Cartilage replacement compositions and methods

ABSTRACT

Described are compositions and methods for cartilage replacement. Also described are collagen scaffolds comprising the composition described herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/826,819, filed Mar. 29, 2019, which is incorporated herein byreference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 19, 2020 isnamed “213474-9001-US02_SequenceListing.txt” and is 1 kb in size.

TECHNICAL FIELD

The present disclosure relates to compositions and methods for cartilagereplacement.

BACKGROUND

While the body has efficient processes for healing and replacing mostdamaged tissue, the tissues in a joint often fail to heal and break downover the lifetime of an individual. In healthy individuals, the end ofbones have a smooth surface made of cartilage. The cartilage islubricated by a thin layer of synovial fluid allowing bones to slidesmoothly against each other but also can prevent tissue healing. Whenthe cartilage deteriorates or is injured, the joint becomes stiff andpainful. One way to restore motion and reduce pain is through jointreplacement surgery that replaces damaged bone with an artificialimplant, usually of coated or uncoated metal or plastic. In addition tolong recovery times and risk associated with any major invasive surgery,the artificial implants can be fraught by dislocation, fracturing, anderosion, which all end up resulting in additional surgical intervention.

A potential alternative to joint replacement exists through theinsertion of new cartilage into the affected joint or a scaffold forgenerating new cartilage in situ. Chondrocytes, cartilage cells, can bereproduced in vitro, but placing the chondrocytes in a particularlocation and ensuring proliferation is challenging. In addition,injecting chondrocytes into a joint fails to recognize the fact thatcartilage is more than just cells. Cartilage tissue is composed mostlyof non-cellular material including water, collagen, and otherextra-cellular matrix materials, most of which are produced and/ormaintained by chondrocytes. Various scaffolds have been suggested tomimic the other components of cartilage including nanofibers, hydrogels,beads, mashes, and microspheres. The use of artificial compounds to formthese scaffolds, however, pose their own clinical problems and adverseside effects. In addition, most of the currently used scaffolds do notaddress a mechanism to adhere the scaffold or replacement cartilage tothe bone.

There remains a need, therefore, for compositions and methods forefficient, long-lasting, and minimally invasive cartilage replacement.

BRIEF SUMMARY

In one aspect, the disclosure provides compositions comprising collagen,an elastin peptide, and a divalent cation.

In another aspect, the disclosure provides a collagen scaffold. Thecollagen scaffold may comprise the composition as disclosed herein and afibrin sealant. The disclosure also provides methods of making thecollage scaffold described herein.

In another aspect, the disclosure provides a method for the replacementof damaged cartilage in a subject. The method may comprise removingdamaged cartilage from the subject, preparing a composition as describedherein, mixing the composition with a fibrin sealant to form a collagenscaffold, and injecting the collagen scaffold into the subject.

In another aspect, the disclosure also provides a kit. The kit comprisescollagen, an elastin peptide, a divalent cation, and a fibrin sealant.

Other aspects and embodiments of the disclosure will become apparent inlight of the following description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A, FIG. 1B, FIG. 1C, and FIG. 1D are images showing theimplantation of the collagen scaffold compositions described herein.FIG. 1A shows the site of implantation as designated by the hashedcircled area. FIG. 1B and FIG. 1C show images taken during implantationof the collagen scaffold compositions described herein. FIG. 1D showsthe adhered collagen scaffold to the bone following implantation.

FIG. 2A, FIG. 2B, and FIG. 2C are images showing the implantation of acontrol collagen scaffold composition comprising type II collagenwithout the elastin peptide or calcium gluconate. FIG. 2A and FIG. 2Bshow images taken during implantation of the control collagen scaffold.FIG. 2C shows the lack of the control collagen scaffold adhered to thebone following implantation.

DETAILED DESCRIPTION

The present disclosure provides compositions and methods for cartilagereplacement. The composition has the ability to incorporate and nurturechondrocyte containing tissues while adhering to the bone. Thecomposition includes collagen, an elastin peptide, and a divalentcation. The composition may be combined with a fibrin sealant or fibringlue, to form a collagen scaffold for use in cartilage replacementmethods.

1. Definitions

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present document, includingdefinitions, will control. Preferred methods and materials are describedbelow, although methods and materials similar or equivalent to thosedescribed herein can be used in practice or testing of the presentinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety. The materials, methods, and examples disclosed herein areillustrative only and not intended to be limiting.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to better illuminate theinvention and does not pose a limitation on the scope of the inventionunless otherwise claimed. No language in the specification, and nostructures shown in the drawings, should be construed as indicating thatany non-claimed element is essential to the practice of the invention.

The terms “comprise(s),” “include(s),” “having,” “has,” “can,”“contain(s),” and variants thereof, as used herein, are intended to beopen-ended transitional phrases, terms, or words that do not precludethe possibility of additional acts or structures. The singular forms“a,” “an” and “the” include plural references unless the context clearlydictates otherwise. The present disclosure also contemplates otherembodiments “comprising,” “consisting of” and “consisting essentiallyof,” the embodiments or elements presented herein, whether explicitlyset forth or not.

The modifier “about” used in connection with a quantity is inclusive ofthe stated value and has the meaning dictated by the context (forexample, it includes at least the degree of error associated with themeasurement of the particular quantity). The modifier “about” shouldalso be considered as disclosing the range defined by the absolutevalues of the two endpoints. For example, the expression “from about 2to about 4” also discloses the range “from 2 to 4.” The term “about” mayrefer to plus or minus 10% of the indicated number. For example, “about10%” may indicate a range of 9% to 11%, and “about 1” may mean from0.9-1.1. Other meanings of “about” may be apparent from the context,such as rounding off, so, for example, “about 1” may also mean from 0.5to 1.4.

For the recitation of numeric ranges herein, each intervening numberthere between with the same degree of precision is explicitlycontemplated. For example, for the range of 6-9, the numbers 7 and 8 arecontemplated in addition to 6 and 9, and for the range 6.0-7.0, thenumber 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 areexplicitly contemplated.

As used herein, the terms “administering,” “providing” and “introducing”are used interchangeably and refer to the placement of the compositionsof the disclosure into a subject by a method or route that results in atleast partial localization of the composition to a desired site. Thecompositions may be administered by any appropriate route that resultsin delivery to a desired location in the subject.

As used herein, the term “amino acid” refers to naturally occurring andnon-natural synthetic amino acids as well as amino acid analogs andamino acid mimetics that function in a manner similar to the naturallyoccurring amino acids. Naturally occurring amino acids are those encodedby the genetic code. Amino acids can be referred to herein by eithertheir commonly known three-letter symbols or by the one-letter symbolsrecommended by the IUPAC-IUB Biochemical Nomenclature Commission. Aminoacids include the side chain and polypeptide backbone portions.

As used herein, the term “divalent cation” or “bivalent cation” are usedto indicate a chemical element in which it has a positive charge stateof two. For example, Ca′ is the divalent cation of calcium.

As used herein, the term “elastin peptide” in either singular or pluralform refers to a peptide or amino acid sequence that corresponds to, isthe biological equivalent of, is analogous with, or is substantiallyhomologous with a portion of elastin but is not full-length elastin. Theterm “elastin peptide” is not meant to convey any meaning regarding thesource or starting material or method of arriving at the elastinpeptide.

As used herein, “fibrin sealant” or “fibrin glue” refers to a surgicalformulation used to create a fibrin clot for hemostasis or woundhealing. The sealants usually include fibrinogen and thrombin, which,when reconstituted in the presence of calcium chloride, convert thefibrinogen into fibrin monomers and give rise to a three-dimentionalgel. Fibrin sealant preparations may also include additional componentsincluding aprotinin, fibronectin, and plasminogen. An example of afibrin sealant includes Beriplast® P sealant from CSL Behring.

A “peptide” or “polypeptide” is a linked sequence of two or more aminoacids linked by peptide bonds. The polypeptide may be natural,synthetic, or a modification or combination of natural and synthetic.Domains are portions of a polypeptide or protein that form a compactunit and are typically 15 to 350 amino acids long.

A “subject” or “patient” may be human or non-human and may include, forexample, animal strains or species including those used as “modelsystems” for research purposes, such a mouse model as described herein.Likewise, patient may include either adults or juveniles (e.g.,children). Moreover, patient may mean any living organism, preferably amammal (e.g., human or non-human) that may benefit from theadministration of compositions contemplated herein. Examples of mammalsinclude, but are not limited to, any member of the Mammalian class:humans, non-human primates such as chimpanzees, and other apes andmonkey species; farm animals such as cattle, horses, sheep, goats,swine; domestic animals such as rabbits, dogs, and cats; laboratoryanimals including rodents, such as rats, mice and guinea pigs, and thelike. Examples of non-mammals include, but are not limited to, birds,fish and the like. In one embodiment of the methods and compositionsprovided herein, the mammal is a human.

2. Composition

The present disclosure provides a composition comprising collagen, anelastin peptide, and a divalent cation.

a. Collagen

The composition may comprise collagen. The collagen may be purified froma number of biological sources known in the art. For example, collagenproteins may be extracted from the connective tissues in the skin, bone,tendon, or other tissues of animals, including humans and bovines, aswell as from fish skin and scale. Purified collagen may be furthertreated for sterilization. Alternatively, the collagen may be producedin vitro using well known molecular biology techniques. The collagen mayhave an amino acid sequence or a modified amino acid sequence of thatfound naturally in animals and fish.

The collagen may be any type of collagen. The collagen may besolubilized collagen. The collagen may form a fibrillar structure. Thecollagen may be Type 1, Type II, Type III, Type IV, Type V or acombination thereof. In some embodiments, the collagen is Type IIcollagen.

The composition may comprise about 1×10⁻⁶ to about 10×10⁻⁶ molescollagen. The composition may comprise at least 1×10⁻⁶ moles, at least2×10⁻⁶ moles, at least 3×10⁻⁶ moles, at least 4×10⁻⁶ moles, at least5×10⁻⁶ moles, at least 6×10⁻⁶ moles, at least 7×10⁻⁶ moles, at least8×10⁻⁶ moles, or at least 9×10⁻⁶ moles collagen. The composition maycomprise less than 10×10⁻⁶ moles, less than 9×10⁻⁶ moles, less than8×10⁻⁶ moles, less than 7×10⁻⁶ moles, less than 6×10⁻⁶ moles, less than5×10⁻⁶ moles, less than 4×10⁻⁶ moles, less than 3×10⁻⁶ moles, or lessthan 2×10⁻⁶ moles collagen. In some embodiments the compositioncomprises about 3×10⁻⁶ moles collagen.

b. Elastin Peptide

The composition may comprise an elastin peptide. The elastin may bederived from sources known in the art. The elastin peptide may beisolated from an enzymatic digestion pool. The elastin peptide may besynthesized with a peptide sequencer. The molecular weight or length ofthe elastin peptide to be used in the present invention is not limited.The elastin peptide does not encompass, however, full-length elastin.

The elastin peptide may comprise an amino acid sequence of XGXXPG (SEQID NO: 1), wherein X is any amino acid. In some embodiments, the elastinpeptide comprises an amino acid sequence of VGVAPG (SEQ ID NO: 2).

The composition may comprise about 1×10⁻⁶ to about 10×10⁻⁶ moles elastinpeptide. The composition may comprise at least 1×10⁻⁶ moles, at least2×10⁻⁶ moles, at least 3×10⁻⁶ moles, at least 4×10⁻⁶ moles, at least5×10⁻⁶ moles, at least 6×10⁻⁶ moles, at least 7×10⁻⁶ moles, at least8×10⁻⁶ moles, or at least 9×10⁻⁶ moles elastin peptide. The compositionmay comprise less than 10×10⁻⁶ moles, less than 9×10⁻⁶ moles, less than8×10⁻⁶ moles, less than 7×10⁻⁶ moles, less than 6×10⁻⁶ moles, less than5×10⁻⁶ moles, less than 4×10⁻⁶ moles, less than 3×10⁻⁶ moles, or lessthan 2×10⁻⁶ moles elastin peptide. In some embodiments, the compositioncomprises about 5×10⁻⁶ moles elastin peptide.

The composition may comprise any absolute quantity of collagen andelastin peptide that results in a mole ratio of collagen to elastinpeptide of about 1:1 to about 1:5. In some embodiments, the compositioncomprises a mole ratio of collagen to elastin peptide of about 1:1.6.

c. Divalent Cation

The composition may comprise a divalent cation. The divalent cation maybe any cation with a positive two charge, for example, zinc, calcium,magnesium, manganese, iron, and copper. In preferred embodiments, thedivalent cation is calcium, magnesium, or a combination thereof.

The divalent cation may comprise an organic or inorganic salt of thedivalent cation. For example, the divalent cation may be joined with anorganic or inorganic anion to form an organic or inorganic salt. Theorganic or inorganic salt may be a chloride salt, a carbonate salt, agluconate salt, a phosphate salt, a sulphate salt, a bicarbonate salt,an acetate salt, a citrate salt, a silicate salt, a pyrophosphate salt,an oxide salt, an oxalate salt, a nitrite salt, a nitrate salt, alactate salt, a hydroxide salt, a glucoheptonate salt, an ascorbatesalt, or a combination or hydrate thereof. In exemplary embodiments, thedivalent cation is calcium gluconate.

The composition may comprise about 0.5 to about 10 millimoles divalentcation. The composition may comprise at least 0.5 millimoles, at least 1millimole, at least 2 millimoles, at least 3 millimoles, at least 4millimoles, at least 5 millimoles, at least 6 millimoles, at least 7millimoles, at least 8 millimoles, or at least 9 millimoles divalentcation. The composition may comprise less than 10 millimoles, less than9 millimoles, less than 8 millimoles, less than 7 millimoles, less than6 millimoles, less than 5 millimoles, less than 4 millimoles, less than3 millimoles, less than 2 millimoles, or less than 1 millimole divalentcation. In some embodiments, the composition comprises about 7millimoles divalent cation.

The composition may comprise any absolute quantity of collagen, elastinpeptide, and divalent cation which results in a mole ratio of collagento elastin peptide to divalent cation of about 1:1:1000 to about1:5:4000. The composition may comprise a mole ratio of collagen toelastin peptide to divalent cation of about 1:1:2000, about 1:1:3000,about 1:1:4000, about 1:2:1000, about 1:2:2000, about 1:2:3000, about1:2:4000, about 1:3:1000, about 1:3:2000, about 1:3:3000, about1:3:4000, about 1:4:1000, about 1:4:2000, about 1:4:3000, about1:4:4000, about 1:5:1000, about 1:5:2000, or about 1:5:3000. In someembodiments, the composition comprises a mole ratio of collagen toelastin peptide to divalent cation of about 1:1.6:2300.

The composition may further comprise a solvent. Suitable solventsinclude water, isotonic saline, or a buffer, for example, phosphate,Tris, HEPES, or other biologically suitable buffer.

The composition may incorporate therapeutic proteins including, but notlimited to, hormones, cytokines, growth factors, clotting factors,anti-protease proteins (e.g., alphal-antitrypsin), angiogenic proteins(e.g., vascular endothelial growth factor, fibroblast growth factors),antiangiogenic proteins (e.g., endostatin, angiostatin), and otherproteins that are present in the blood, bone morphogenic proteins(BMPs), osteoinductive factor (IFO), fibronectin (FN), endothelial cellgrowth factor (ECGF), cementum attachment extracts (CAE), ketanserin,human growth hormone (HGH), animal growth hormones, epidermal growthfactor (EGF), interleukin-1 (IL-1), human alpha thrombin, transforminggrowth factor (TGF-beta), insulin-like growth factor (IGF-1), plateletderived growth factors (PDGF), fibroblast growth factors (FGFs), andperiodontal ligament chemotactic factor (PDLGF), for therapeuticpurposes.

3. Collagen Scaffold

The present disclosure provides a collagen scaffold comprising thecompositions described herein and a fibrin sealant.

The fibrin sealant may be any fibrin sealant known in the art.Typically, a fibrin sealant is formed by enzymatic reactions involvingfibrinogen and thrombin. The fibrin sealant may comprise amulti-component device, such that two supply reservoirs, or two syringesare used to keep the fibrinogen and thrombin separate untiladministration. The fibrin sealant may comprise a single-componentdevice.

The present disclosure also provides a method of making a collagenscaffold, comprising preparing a composition as described herein andmixing the composition with a fibrin sealant. The compositions describedherein may be mixed with all or part of the fibrin sealant. For example,if a multi-component device for the fibrin sealant is used, thecomposition may be mixed with one or both of the fibrinogen and thrombinbefore administration.

The collagen scaffold may additionally serve as a base to add elementsthat can help repair cartilage damage. For example, these elements mayinclude cellular components, including but not limited to, progenitorcells, chondrocytes, either autologous, homologous, or heterologous ornon-cellular components, such as endosomes, exosomes, vacuoles or anyother element useful in the repair of cartilage lesions.

4. Methods of Use

The present disclosure also provides a method for the replacement ofdamaged cartilage in a subject. The method comprises: removing damagedcartilage from the subject, preparing a composition as described herein,mixing the composition with a fibrin sealant to form a collagenscaffold, and injecting the collagen scaffold into the subject toreplace the damaged cartilage.

Removing the damaged cartilage also includes locating and identifyingdamaged cartilage in the subject. This may be done, for example, withmagnetic resonance imaging. The cartilage may be removed by anywell-known method in the art. The surgeon can recognize the best andmost applicable method to use based on the size and location of thedamaged cartilage. The surgery may involve a standard synovectomy,bursectomy, or capsulotomy. The cartilage may be removed by the use ofshavers, basket tweezers, or chondrotomic or other suitable devices.

If the underlying bone does not have a normal surface, bone formationwithin the cartilage, endostosis, or new bone on the surface of thebone, exostosis, has to be removed and repaired with osseous matrixprior to injection of the collagen scaffold.

Following mixing the compositions described herein with the fibrinsealant to form a collagen scaffold, the collagen scaffold is injectedinto the area in which the damaged cartilage was removed. The injectionmay be completed in the absence of water.

The collagen scaffolds described herein may be used alone or incombination with other three-dimensional (3-D) scaffolds or othertraditional repair devices or techniques.

5. Kit

The present disclosure additionally provides kits comprising:

-   -   (a) collagen;    -   (b) an elastin peptide;    -   (c) a divalent cation; and    -   (d) a fibrin sealant.

In some embodiments, the collagen, the elastin peptide, and the divalentcation are co-formulated. In some embodiments, the collagen, the elastinpeptide, and the divalent cation are co-formulated with the fibrinsealant. In some embodiments, the collagen, the elastin peptide, and thedivalent cation, and the fibrin sealant are co-packaged.

The kits can also comprise compounds and/or products co-packaged,co-formulated, and/or co-delivered with other components. For example, adrug manufacturer, a physician, or a hospital can provide a kitcomprising those components listed above and another component fordelivery to a patient.

The disclosed kits can be employed in connection with disclosed methods.

The kits may further include information, instructions, or both on howthe kit can be used to repair damaged cartilage. The information andinstructions may be in the form of words, pictures, both, or the like.

6. Examples

It will be readily apparent to those skilled in the art that othersuitable modifications and adaptations of the methods of the presentdisclosure described herein are readily applicable and appreciable andmay be made using suitable equivalents without departing from the scopeof the present disclosure or the aspects and embodiments disclosedherein. Having now described the present disclosure in detail, thedisclosure will be more clearly understood by reference to the followingexamples, which are merely intended only to illustrate some aspects andembodiments of the disclosure, and should not be viewed as limiting tothe scope of the disclosure. The disclosures of all journal references,U.S. patents, and publications referred to herein are herebyincorporated by reference in their entireties.

Example 1. Preparation of the Composition

0.5 mLs of a solution containing elastin peptide at 10.6 mM (GrupoProteo) was mixed with 2 mLs of bovine Type 2 collagen 1.5 mM and 0.5mLs calcium gluconate (PiSA® Farmaceutica) at a concentration of 14 M inan appropriate solvent.

The elastin peptide was hydrolyzed peptides of the full-length elastinprotein, with an average molecular weight of 7000 Daltons. The averagemolecular weight was used to calculate the concentration recited above.

By nature, Type 2 collagen is usually hydrophilic and fails to work in ascaffold. When mixed with the elastin peptide and divalent cation,however, it was surprisingly found that the type II collagen takes on ahydrophobic nature. This surprising hydrophobicity made the compositionappropriate for population with chondrocytes and/or chondrocytecontaining tissues.

Example 2. Preparation of the Collagen Scaffold

In addition to hydrophobic type 2 collagen, elastin peptide and calciumgluconate, the scaffold contains a fibrin sealant. An example of theinvention used the commercially available Beriplast® P sealant. TheBeriplast® P is a two-component sealant in which one syringe contains 2mL of a solution of fibrinogen and aprotinin and a second syringe with asolution of calcium chloride and thrombin. The composition was mixedwith the thrombin solution of the second syringe. In addition, a fibrinsealant can be used that does not include aprotinin. Omitting aprotininfrom the fibrin sealant can improve the strength of the scaffold.

Example 3. Cartilage Replacement

The formulated scaffold as described in Example 2 was used for acartilage replacement surgery. The damaged cartilage was located bymeans of CartiloGram magnetic resonance imaging. Two portals in the skinwere opened by way of an ordinary arthroscopic surgery. Further imagingof the cartilage damage was observed after insertion of a trocar with acamera through one of the portals.

An initial standard synovectomy, bursectomy, or capsulotomy wascompleted and the damaged cartilage, which is relatively softer than thenormal cartilage, was removed by shavers, backet tweezers, and/orchondrotomic devices. The removal of the damaged cartilage was verifiedby a stylus hook and touch. In some instances, thermal radio frequencywas also used to define the edge between normal and damaged cartilage.

After confirmation that the bone had a normal surface, the fibrinsealant was mixed with the composition as described in Example 2 to formthe collage scaffold and injected into the lesion. A hook stylus wasused to help the scaffold stick to the bone in regular form. During theinjection of the scaffold, the flow of water was stopped for tenseconds. After two minutes, the behavior of the solidified scaffold waschecked by observing joint movement.

The use of the material was also tested in a porcine model, as shown inFIGS. 1A-1D. A site for implantation was designated (FIG. 1A, hashedcircle). During implantation of the material described herein, thematerial was injected and remained near the site for implantation (FIG.1B). Following injection, a hook stylus was used to shape the scaffoldaround the site of implantation (FIG. 1C). As shown in FIG. 1D, thescaffold adhered to the bone in the site of implantation and formed aregular scaffold structure.

For comparison, a control scaffold only containing type II collagenwithout the elastin peptide or the calcium gluconate was also preparedand tested in the porcine model (FIGS. 2A-2C). During implantation, thecontrol scaffold did not remain near the site for implantation and wasunable to adhere to the bone throughout the process (FIG. 2A and FIG.2B). As shown in FIG. 2C, following injection, only a small amount ofthe control scaffold remained at the site of implantation. Even thatsmall amount was unable to effectively adhere to the bone.

The scaffold compositions described herein are able to be easilyimplanted using standard orthoscopic techniques and implements. Contraryto the control scaffold, the scaffold compositions described hereinproduced a scaffold which showed hydrophobic characteristics such thatwhen injected it did not diffuse away from the site of implantation. Inaddition, the scaffold composition showed a superior ability to adhereto the bone at the site of implantation and offer a more stable, longerlasting scaffold for cartilage repair.

For reasons of completeness, various aspects of the invention are setout in the following numbered clauses:

Clause 1. A composition comprising collagen, an elastin peptide, and adivalent cation.

Clause 2. The composition of clause 1, wherein the collagen is type 2collagen.

Clause 3. The composition of clause 1 or clause 2, wherein thecomposition comprises about 1×10⁻⁶ to about 10×10⁻⁶ moles collagen.

Clause 4. The composition of any of clauses 1-3, wherein the compositioncomprises about 3×10⁻⁶ moles collagen.

Clause 5. The composition of any of clauses 1-4, wherein the elastinpeptide comprises an amino acid sequence of XGXXPG (SEQ ID NO: 1),wherein X is any amino acid.

Clause 6. The composition of any of clauses 1-5, wherein the elastinpeptide comprises an amino acid sequence of VGVAPG (SEQ ID NO: 2).

Clause 7. The composition of any of clauses 1-6, wherein the compositioncomprises about 1×10⁻⁶ to about 10×10⁻⁶ moles elastin peptide.

Clause 8. The composition of any of clauses 1-7, wherein the compositioncomprises about 5×10⁻⁶ moles elastin peptide.

Clause 9. The composition of any of clauses 1-8, wherein the compositioncomprises a mole ratio of collagen to elastin peptide of about 1:1 toabout 1:5.

Clause 10. The composition of any of clauses 1-9, wherein the divalentcation is magnesium or calcium.

Clause 11. The composition of any of clauses 1-10, wherein the divalentcation comprises an organic or inorganic salt.

Clause 12. The composition of any of clauses 1-11, wherein the organicor inorganic salt is a chloride salt, a carbonate salt, a gluconatesalt, a phosphate salt, a sulphate salt, a bicarbonate salt, an acetatesalt, a citrate salt, a silicate salt, a pyrophosphate salt, an oxidesalt, an oxalate salt, a nitrite salt, a nitrate salt, a lactate salt, ahydroxide salt, a glucoheptonate salt, an ascorbate salt, or acombination or hydrate thereof.

Clause 13. The composition of any of clauses 1-12, wherein the divalentcation is calcium gluconate.

Clause 14. The composition of any of clauses 1-13, wherein thecomposition comprises about 0.5 to about 10 millimoles divalent cation.

Clause 15. The composition of any of clauses 1-14, wherein thecomposition comprises about 7 millimoles divalent cation.

Clause 16. The composition of any of clauses 1-15, wherein thecomposition comprises a mole ratio of collagen to elastin peptide todivalent cation of about 1:1:1000 to about 1:5:4000.

Clause 17. A collagen scaffold comprising: the composition of any ofclauses 1-16 and a fibrin sealant.

Clause 18. A method of making a collagen scaffold, comprising: preparinga composition of any of clauses 1-16 and mixing the composition with afibrin sealant.

Clause 19. A method for the replacement of damaged cartilage in asubject in need thereof, comprising: removing damaged cartilage from thesubject; preparing a composition of any of clauses 1-16; mixing thecomposition with a fibrin sealant to form a collagen scaffold; andinjecting the collagen scaffold into the subject to replace the damagedcartilage.

Clause 20. A kit comprising: collagen; an elastin peptide; a divalentcation; and a fibrin sealant.

The invention claimed is:
 1. A composition comprising: collagen type II;an elastin peptide; and calcium gluconate.
 2. The composition of claim1, wherein the composition comprises about 1×10⁻⁶ to about 10×10⁻⁶ molesof collagen type II.
 3. The composition of claim 1, wherein thecomposition comprises about 3×10⁻⁶ moles of collagen type II.
 4. Thecomposition of claim 1, wherein the elastin peptide comprises an aminoacid sequence of XGXXPG (SEQ ID NO: 1), wherein X is any amino acid. 5.The composition of claim 1, wherein the elastin peptide comprises anamino acid sequence of VGVAPG (SEQ ID NO: 2).
 6. The composition ofclaim 1, wherein the composition comprises about 1×10⁻⁶ to about 10×10⁻⁶moles elastin peptide.
 7. The composition of claim 6, wherein thecomposition comprises about 5×10⁻⁶ moles elastin peptide.
 8. Thecomposition of claim 1, wherein the composition comprises a mole ratioof collagen type II to elastin peptide of about 1:1 to about 1:5.
 9. Thecomposition of claim 1, wherein the composition comprises about 0.5 toabout 10 millimoles of calcium gluconate.
 10. The composition of claim9, wherein the composition comprises about 7 millimoles of calciumgluconate.
 11. The composition of claim 1, wherein the compositioncomprises a mole ratio of collagen type II to elastin peptide to calciumgluconate of about 1:1:1000 to about 1:5:4000.
 12. A collagen scaffoldcomprising: the composition of claim 1 and a fibrin sealant.
 13. A kitcomprising: collagen type II; an elastin peptide; calcium gluconate; anda fibrin sealant.
 14. A method of making a collagen scaffold,comprising: preparing the composition of claim 1, and mixing thecomposition with a fibrin sealant to produce a collagen scaffold.
 15. Amethod for the replacement of damaged cartilage in a subject in needthereof, comprising: removing damaged cartilage from the subject; mixingthe composition of claim 1 with a fibrin sealant to form a collagenscaffold; and administering the collagen scaffold into the subject toreplace the damaged cartilage.